The effects of tangerine (Phaseolus vulgaris Blanco), lemon (Citrus medica limonum Lush), pear orange (Citrus sinensis L. Osbeck), red copaiba (Copaifera langsdorffii Desf.), rosemary (Baccharis dracunculifolia De Candole), Eucalyptus (Eucalyptus globulus Labillardière and E. citriodora Hook), lemongrass (Cymbopogon citratus Stapf.) and citronella (Cimbopogon nardus Linnaeus) oils at several concentrations on Zabrotes subfasciatus (Boheman) were studied. In toxicity tests, grains of Phaseolus vulgaris L. cv. Rajadinho were impregnated with oils and infested with adults of Z. subfasciatus up to 24 hours old. All tested oils were effective in reducing the viable egg-laying and adult emergence of this pest, in function of the concentrations used, highlighting E. citriodora and E. globulus oils which caused 100% effectiveness from 0.5 mL Kg-1 concentration. In repellency tests, two arenas consisting of plastic containers, connected symmetrically to a central box by two plastic tubes were used. In one of the boxes, untreated beans were placed and on the other ones beans treated with each oil concentration were used. In the central box, five couples of Z. subfasciatus were released. Grains of P. vulgaris treated with oils of E. citriodora, C. citratus and C. oleifera reduced the attraction percentage of Z. subfasciatus adults, while the E. globulus increased this percentage. The percentages of reduced viable eggs ranged from 17.9% (C. medica limonum) to 93.3% (C. nardus), while the reduction on the number of emerged insects was 23.9% and 95.9%, respectively for these same oils.

The Bruchinae are distributed in almost all continents except in Antarctica and New Zealand, being the largest number of species found in the Neotropical Region (Kingsolver 2002). The species of stored products may have originated in various regions of the world, but due to transport from one country or continent to another together with the goods they have become cosmopolitan. It is believed that many of these species occur in populations, geographic subspecies or breeds that differ in food preference, climatic conditions and susceptibility to insecticides (Hill 1990).

Zabrotes subsfasciatus (Boheman) (Coleoptera, Chrysomelidae, Bruchinae) is a major primary pest of the stored bean Phaseolus vulgaris L. in tropical regions. It infests grains and seeds in storage units causing quantitative losses (weight loss), which can be quite variable depending on the degree of infestation and susceptibility of seeds or grains from such species. Qualitative losses are more difficult to be assessed, and they consist of vigor and germination reduction, changes in physical appearance, nutritional losses, presence of insects, mites and their fragments, fungal infection and mycotoxin contamination, impurities and other foreign materials (Lazzari 1997).

Botanical insecticides containing different compounds derived from plants secondary metabolism have been tested in order to control this pest in stored beans with promising results (Weaver et al. 1994; Oliveira and Vendramim 1999; Barbosa et al. 2002; Paranhos et al. 2005, Santos et al. 2007; Silva et al. 2008; Paul et al. 2009), as an alternative to protective chemical insecticides, which are generally more toxic and likely to cause harmful effects to applicators, consumers and the environment. Among the most promising compounds, the monoterpenes and its analogues are remarkable. They are quite volatile, lipophilic and act by contact, ingestion and fumigation, causing mortality, repellence and effects on the insect biology, physiology and behavior. They can be used as dry powders, extracts, essential oils and oil emulsion (Brito et al. 2006; Isman 2006; Rajendran and Sriranjini 2008; Selase and Getu 2009).

This study aimed to evaluate the effects of some essential oils on mortality, number of viable eggs and emergence of Z. subfasciatus, as well as its repellency to adults on grains of P. vulgaris.

MATERIALS AND METHODS

Zabrotes subfasciatus rearing. Insects were reared for several generations on grains of P. vulgaris cv. Rajadinho in the Agricultural Entomology Laboratory of the Federal Rural University of Pernambuco (UFRPE), Brazil, at 27.1 ± 1 ºC, 74.2 ± 5.3% of RH (Humidity Relative) and 12 h of photophase, stored in glass containers, sealed with perforated plastic lids and lined with fine cloth to allow gas exchange. Adults were confined for six days, subsequently they were removed, and the grains stored until adult emergence.

Infestation removal and hygroscopic equilibrium. Beans properly selected (broken grains with impurities were eliminated) used for rearing and experiments were placed in plastic bags and kept in a freezer at -5 ºC for seven days to eliminate possible insect infestation from field. After removal from the freezer, the grains were transferred to glass flasks and kept in laboratory temperature, which varied between 26 and 28 oC for seven days in order to reach the hygroscopic equilibrium. The equilibrium with air humidity is normally reached after seven days.

Contact toxicity tests. Experiments were conducted at 30.1 ± 1.8 ºC, 67.2 ± 4.7% Relative Humidity and 12 h photophase. Oils were tested at the concentrations 0, 0.5, 1.0, 1.5, 2.0 and 2.5 mL Kg-1 grains of P. vulgaris cv. Rajadinho, selected in preliminary experiments. The treatment of grains with the essential oils was performed using plastic containers. Oils were manipulated using an automatic pipette and then the containers were manually agitated during two minutes. Each 20 g portion of P. vulgaris grains was infested with five couples of Z. subfasciatus up to 24 h old. Six days after the confinement, live and dead insects were counted and then discarded. The quantification of viable and unviable eggs was performed after 16 days, examining all grains of each plot under stereomicroscope. The viable eggs are opaque whereas the non-viable are translucent. Adult emergences were measured at 40 days after infestation. The oils were separately tested in a fully randomized design, consisting of six treatments and six replications. Results were submitted to regression analysis, being selected those equations that best represented the biological response, based on its significance (F and P) and higher coefficient of determination (R2). All tests were conducted using the statistical program SAS version 8.02 (SAS Institute 2001).

Repellency tests. The same oils used above were studied. The experiments were conducted at the same range of temperature, relative humidity and photoperiod. Arenas consisting of two plastic containers, connected symmetrically to a central box by two plastic tubes were used. In one of the boxes, 20 g of beans without oil (control) were placed and on the other one, the same amount of treated grains was used. In the central box five couples of Z. subfasciatus up to 24 h old were released. The oils were tested separately in a complete randomized design, consisting of two treatments: the best oil concentration in the no-choice test and the control, using 10 replicates. After six days, insects maintained in each container were counted for repellency evaluation, and then discarded; the beans were stored in plastic containers. The viable and unviable eggs were evaluated in all grains of the plot after 16 days, and emerged adults quantified at 40 days after infestation.

Percentages of adults attracted to the control and to each concentration of oil were submitted to the analysis of choice frequency and evaluated by the chi-square test using the SAS. The number of viable eggs and the number of emerged insects were subjected to analysis of variance; data from B. dracunfolia, C. citratus, C. langsdorffii, C. nardus e C. reticulata were transformed into square root of x +0.5, in order to achieve homogeneity in variances, and analyzed by the t test. The average percentage reduction of oviposition and adult emergence was calculated using the formula adapted from Obeng-Ofori (1995): PR = [(NC - NT) / (NC + TN) x 100], where PR = average percentage of reduction in oviposition and/or emergence, NC = average number of eggs and/or emerged insects in the control, and NT = average number of eggs and/or emerged insects in the treatment.

RESULTS AND DISCUSSION

Contact toxicity tests. Mortality rates of Z. subfasciatus on grains of P. vulgaris treated with plant essential oils varied with both oil type and concentration used. Regression equations (significant, P ≤ 0.05) that explain oils performance on adult mortality, number of viable eggs, and emerged adults were obtained (Table 1). No significant regression equations were established for the essential oils of E. globulus, E. citriodora, C. nardus, C. citratus, B. drancunculifolia and C. sinensis, as all of them caused 100% mortality rates and total reduction on the number of viable eggs and emerged insects from the lowest concentration (0.5 mL Kg-1 beans) not fitting the statistical model. Although presenting 100% efficiency, the results demonstrated the need to test these oils at lower concentrations, for the establishment of regression equations.

In Brazil there are only four products registered for control of Z. subfasciatus in stored beans, being three inorganic precursors of phosphine fumigants (extremely toxic), used to curative control, and a pyrethroid (deltamethrin), as a protective insecticide (AGROFIT 2011). Hence, the results of this work, with essential oils that may features less impact on environment and health of applicators and consumers, are crucial as alternative products.

The fumigant effect of essential oils has been also investigated, opening new perspectives in the control of stored grain pests with plants of the families Apiaceae, Lamiaceae, Lauraceae and Myrtaceae, aiming mainly to control immature and adults of Tribolium castaneum Herbst, Rhyzopertha dominica Fabricius, Sitophilus oryzae Linnaeus and Sitophilus zeamais Motschulsky (Rajendran and Sriranjini 2008), and that can be tested for the management of Z. subfasciatus. Essential oils from plants of the genus Eucalyptus reduced the proportion of viable eggs and emerged adults of Z. subfasciatus due to its fumigant effect (Brito et al. 2006). The essential oil from leaves and stems of Croton grewioides Baill. (Euphorbiaceae) also presented fumigant effect to adults of Z. subfasciatus, with LC50 values of 4 and 13.77µl/L air, respectively (Silva et al. 2008).

The repellency is an important feature of both plant essential oils and powders for the management of Z. subfasciatus, since they play a role directly in the reduction of egg-laying and hence the emergence of adults, making difficult the establishment of pest populations in grain storage units. The oils of cinnamon leaves and bay leaves (Laurus nobilis L.) at 2.5 mL Kg-1 grains of P. vulgaris exerted repellent effect over 70% on adults of Z. subfasciatus (Oliveira and Vendramim 1999). Essential oils from Lippia origanoides Kunth, E. citriodora, and Tagetes lucida Cav. collected in Colombia caused repellency to S. zeamais (Nerio et al. 2009). Oils extracted from Laurus nobilis L., Citrus bergamia (Risso) Wight & Arn., Foeniculum vulgare Mill., and Lavandula hibrida L. were repellent to adults of S. zeamais, Cryptolestes ferrugineus Stephens, and larvae of Tenebrio molitor L. (Cosimi et al. 2009). Nerio et al. (2010) published a recent review about repellency activity of essential oils. This author noted that the compounds of Cymbopogon spp., Ocimum spp. and Eucalyptus spp. were the most repellent to several stored product pests. Due to the own insecticidal properties, plant essential oils can be very useful in integrated management of Z. subfasciatus in stored beans, mainly in small farms, as a promising alternative to synthetic chemical insecticides. However, further studies are a priority regarding to the collecting processes, drying, storage and preparation of plant material, quantification of bioactive compounds, toxicology studies, formulations, and safety. The main goal is to provide consumers with high-quality beans without the presence of toxic waste.